The present invention is in the field of nucleic acid amplification, and specifically in the area of reducing non-specific amplification in nucleic acid amplification reactions.
Numerous nucleic acid amplification techniques have been devised, including strand displacement cascade amplification (SDCA)(referred to herein as exponential rolling circle amplification (ERCA)) and rolling circle amplification (RCA)(U.S. Pat. No. 5,854,033; PCT Application No. WO 97/19193; Lizardi et al., Nature Genetics 19(3):225-232 (1998)); multiple displacement amplification (MDA)(PCT Application WO 99/18241); strand displacement amplification (SDA)(Walker et al., Nucleic Acids Research 20:1691-1696 (1992), Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396 (1992)); polymerase chain reaction (PCR) and other exponential amplification techniques involving thermal cycling, self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), and amplification with Qxcex2 replicase (Birkenmeyer and Mushahwar, J. Virological Methods 35:117-126 (1991); Landegren, Trends Genetics 9:199-202 (1993)); and various linear amplification techniques involving thermal cycling such as cycle sequencing (Craxton et al., Methods Companion Methods in Enzymology 3:20-26 (1991)).
Rolling Circle Amplification (RCA) driven by DNA polymerase can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (Lizardi et al., Nature Genet. 19: 225-232 (1998); U.S. Pat. Nos. 5,854,033 and 6,143,495; PCT Application No. WO 97/19193). If a single primer is used, RCA generates in a few minutes a linear chain of hundreds or thousands of tandemly-linked DNA copies of a target that is covalently linked to that target. Generation of a linear amplification product permits both spatial resolution and accurate quantitation of a target. DNA generated by RCA can be labeled with fluorescent oligonucleotide tags that hybridize at multiple sites in the tandem DNA sequences. RCA can be used with fluorophore combinations designed for multiparametric color coding (PCT Application No. WO 97/19193), thereby markedly increasing the number of targets that can be analyzed simultaneously. RCA technologies can be used in solution, in situ and in microarrays. In solid phase formats, detection and quantitation can be achieved at the level of single molecules (Lizardi et al., 1998). Ligation-mediated Rolling Circle Amplification (LM-RCA) involves circularization of a probe molecule hybridized to a target sequence and subsequent rolling circle amplification of the circular probe (U.S. Pat. Nos. 5,854,033 and 6,143,495; PCT Application No. WO 97/19193).
Artifactsxe2x80x94that is, unwanted, unexpected, or non-specific nucleic acid moleculesxe2x80x94have been observed in almost all nucleic acid amplification reactions. For example, Stump et al., Nucleic Acids Research 27:4642-4648 (1999), describes nucleic acid artifacts resulting from an illegitimate PCR process during cycle sequencing. In rolling circle amplification, uncircularized open circle probes could prime synthesis during amplification of circularized open circle probes. Other forms of artifacts can occur in other types of nucleic acid amplification techniques.
Therefore, it is an object of the present invention to provide a method of reducing, preventing, or eliminating artifacts in nucleic acid amplification reactions.
It is another object of the present invention to provide open circle probes and primers that, when used in a nucleic acid amplification reaction, can reduce, prevent, or eliminate artifacts in the nucleic acid amplification reaction.
It is another object of the present invention to provide kits for nucleic acid amplification that can reduce, prevent, or eliminate artifacts in the nucleic acid amplification reaction.
Disclosed are compositions and methods for reducing or eliminating generation of unwanted, undesirable, or non-specific amplification products in nucleic acid amplification reactions. One form of composition is an open circle probe that can form an intramolecular stem structure, such as a hairpin structure, at one or both ends. Open circle probes are useful in rolling circle amplification techniques. The stem structure allows the open circle probe to be circularized when hybridized to a legitimate target sequence but results in inactivation of uncircularized open circle probes. This inactivation, which preferably involves stabilization of the stem structure, extension of the end of the open circle probe, or both, reduces or eliminates the ability of the open circle probe to prime nucleic acid synthesis or to serve as a template for rolling circle amplification.
In ligation-mediated rolling circle amplification, a linear DNA molecule, referred to as an open circle probe or padlock probe, hybridizes to a target sequence and is circularized. The circularized probe is then amplified via rolling circle replication of the circular probe. Uncircularized probe that remains in the reaction can hybridize to nucleic acid sequences in the reaction and cause amplification of undesirable, non-specific sequences. The disclosed compositions and method address this problem by reducing or eliminating the potential uncircularized open circle probes from priming nucleic acid synthesis. A basic form of the disclosed method involves use of the disclosed open circle probes in a rolling circle amplification reaction or assay.
The disclosed open circle probes can be inactivated in several ways. For example, where the 3xe2x80x2 end of an open circle probe is involved in an intramolecular stem structure, the 3xe2x80x2 end can be extended in a replication reaction using the open circle probe sequences as template. Stabilization of the stem structure results in a reduction or elimination of the ability of the open circle probe to prime nucleic acid synthesis because the 3xe2x80x2 end is stably hybridized to sequences in the open circle probe under the conditions used for nucleic acid replication. The open circle probe can also be inactivated by formation of the intramolecular stem structure during the amplification reaction. As long as the end remains in the intramolecular stem structure, it is not available for priming nucleic acid synthesis. A preferred form of open circle probe includes a loop as part of the intramolecular stem structure. Hybridization of the loop to the target sequence disrupts the intramolecular stem structure while hybridization of the loop to a mismatched or non-target sequence will not. Thus, the sequence-discrimination ability of the open circle probe determines inactivation of the open circle probe. A hybridization nucleating loop can also be used in linear primers used for nucleic acid replication and amplification.
The disclosed method is useful for detection, quantitation, and/or location of any desired analyte, such as proteins and peptides. The disclosed method can be multiplexed to detect numerous different analytes simultaneously or used in a single assay. Thus, the disclosed method is useful for detecting, assessing, quantitating, profiling, and/or cataloging gene expression and the presence of nucleic acids and protein in biological samples. The disclosed method is also particularly useful for detecting and discriminating single nucleotide differences in nucleic acid sequences. Thus, the disclosed method is useful for extensive multiplexing of target sequences for sensitive and specific detection of the target sequences themselves or analytes to which the target sequences have been associated. The disclosed method is applicable to numerous areas including, but not limited to, analysis of proteins present in a sample (for example, proteomics analysis), disease detection, mutation detection, protein expression profiling, RNA expression profiling, gene discovery, gene mapping (molecular haplotyping), agricultural research, and virus detection.